In vitro nucleic acid synthesis is a foundation of many fundamental research and diagnostic tools, such as nucleic acid amplification and sequencing. In a template-dependent nucleic acid synthesis reaction, the sequential addition of nucleotides is catalyzed by a nucleic acid polymerase. Depending on the template and the nature of the reaction, the nucleic acid polymerase may be a DNA polymerase, an RNA polymerase, or a reverse transcriptase. DNA synthesis requires a 3′ hydroxyl on the primer for addition at the α-phosphate of a free deoxynucleotide triphosphate (dNTP). A phosphodiester bond between the nucleotide on the 3′ terminus of the primer and the free nucleotide is formed and diphosphate is released. This reaction is shown schematically in FIG. 1.
The fidelity of template-dependent nucleic acid synthesis depends in part on the ability of the polymerase to discriminate between complementary and non-complementary nucleotides. Normally, the conformation of the polymerase enzyme favors incorporation of the complementary nucleotide. However, there is still an identifiable rate of misincorporation that depends upon factors such as local sequence and the base to be incorporated.
Synthetic or modified nucleotides and analogs, such as labeled nucleotides, tend to be incorporated into a primer less efficiently than naturally-occurring dNTPs. The reduced efficiency with which the unconventional nucleotides are incorporated by the polymerase can adversely affect the performance of sequencing techniques that depend upon faithful incorporation of such unconventional nucleotides.
Single molecule sequencing techniques allow the evaluation of individual nucleic acid molecules in order to identify changes and/or differences affecting genomic function. In single molecule techniques, individual, optically-resolvable nucleic acid fragments are attached to a solid support, and sequencing is conducted on the individual strands. Sequencing events are detected and correlated to the individual strands. See Braslavsky et al., Proc. Natl. Acad. Sci., 100: 3960-64 (2003), incorporated by reference herein. Because single molecule techniques do not rely on ensemble averaging as do bulk techniques, errors due to misincorporation can have a significant deleterious effect on the sequencing results. The incorporation of a nucleotide that is incorrectly paired, under standard Watson and Crick base-pairing, with a corresponding template nucleotide during primer extension may result in sequencing errors. The presence of misincorporated nucleotides may also result in prematurely terminated strand synthesis, reducing the number of template strands for future rounds of synthesis, and thus reducing the efficiency of sequencing.
There is, therefore, a need in the art for improved methods for reducing the frequency of misincorporation and improving fidelity in nucleic acid synthesis reactions, especially in single molecule sequencing.